Highlights d Neuronal ADP:ATP is not affected by neurotransmission despite strong Na + pumping d Stimulated mitochondrial pyruvate flux precedes glucose consumption d Energy stability is not explained by conventional homeostasis (ADP:ATP or Ca 2+) d Na + pump activity controls glycolysis and mitochondrial ATP production

The present work was done to elucidate whether hemichannels of a cell line derived from endothelial cells are affected by pro-inflammatory conditions (high glucose and IL-1β/TNF-α) known to lead to vascular dysfunction. We used EAhy 926 cells treated with high glucose and IL-1β/TNF-α. The hemichannel activity was evaluated with the dye uptake method and was abrogated with selective inhibitors or knocking down of hemichannel protein subunits with siRNA. Western blot analysis, cell surface biotinylation, and confocal microscopy were used to evaluate total and plasma membrane amounts of specific proteins and their cellular distribution, respectively. Changes in intracellular Ca2+ and nitric oxide (NO) signals were estimated by measuring FURA-2 and DAF-FM probes, respectively. High glucose concentration was found to elevate dye uptake, a response that was enhanced by IL-1β/TNF-α. High glucose plus IL-1β/TNF-α-induced dye uptake was abrogated by connexin 43 (Cx43) but not pannexin1 knockdown. Furthermore, Cx43 hemichannel activity was associated with enhanced ATP release and activation of p38 MAPK, inducible NO synthase, COX2, PGE2 receptor EP1, and P2X7/P2Y1 receptors. Inhibition of the above pathways prevented completely the increase in Cx43 hemichannel activity of cells treated high glucose and IL-1β/TNF-α. Both synthetic and endogenous cannabinoids (CBs) also prevented the increment in Cx43 hemichannel opening, as well as the subsequent generation and release of ATP and NO induced by pro-inflammatory conditions. The counteracting action of CBs also was extended to other endothelial alterations evoked by IL-1β/TNF-α and high glucose, including increased ATP-dependent Ca2+ dynamics and insulin-induced NO production. Finally, inhibition of Cx43 hemichannels also prevented the ATP release from endothelial cells treated with IL-1β/TNF-α and high glucose. Therefore, we propose that reduction of hemichannel activity could represent a strategy against the activation of deleterious pathways that lead to endothelial dysfunction and possibly cell damage evoked by high glucose and pro-inflammatory conditions during cardiovascular diseases.

Diverse studies have suggested that cytoplasmic inclusions of misfolded α‐synuclein in neuronal and glial cells are main pathological features of different α‐synucleinopathies, including Parkinson's disease and dementia with Lewy bodies. Up to now, most studies have focused on the effects of α‐synuclein on neurons, whereas the possible alterations of astrocyte functions and neuron–glia crosstalk have received minor attention. Recent evidence indicates that cellular signaling mediated by hemichannels and pannexons is critical for astroglial function and dysfunction. These channels constitute a diffusional route of communication between the cytosol and the extracellular space and during pathological scenarios they may lead to homeostatic disturbances linked to the pathogenesis and progression of different diseases. Here, we found that α‐synuclein enhances the opening of connexin 43 (Cx43) hemichannels and pannexin‐1 (Panx1) channels in mouse cortical astrocytes. This response was linked to the activation of cytokines, the p38 MAP kinase, the inducible nitric oxide synthase, cyclooxygenase 2, intracellular free Ca2+ concentration ([Ca2+]i), and purinergic and glutamatergic signaling. Relevantly, the α‐synuclein‐induced opening of hemichannels and pannexons resulted in alterations in [Ca2+]i dynamics, nitric oxide (NO) production, gliotransmitter release, mitochondrial morphology, and astrocyte survival. We propose that α‐synuclein‐mediated opening of astroglial Cx43 hemichannels and Panx1 channels might constitute a novel mechanism involved in the pathogenesis and progression of α‐synucleinopathies.

The formation of gap junctions was initially thought to be the central role of connexins, however, recent evidence had brought to light the high relevance of unopposed hemichannels as an independent mechanism for the selective release of biomolecules during physiological and pathological conditions. In the healthy brain, the physiological opening of astrocyte hemichannels modulates basal excitatory synaptic transmission. At the other end, the release of potentially neurotoxic compounds through astroglial hemichannels and pannexons has been insinuated as one of the functional alterations that negatively affect the progression of multiple brain diseases. Recent insights in this matter have suggested encannabinoids (eCBs) as molecules that could regulate the opening of these channels during diverse conditions. In this review, we discuss and hypothesize the possible interplay between the eCB system and the hemichannel/pannexon-mediated signaling in the inflamed brain and during event of synaptic plasticity. Most findings indicate that eCBs seem to counteract the activation of major neuroinflammatory pathways that lead to glia-mediated production of TNF-α and IL-1β, both well-known triggers of astroglial hemichannel opening. In contrast to the latter, in the normal brain, eCBs apparently elicit the Ca2+-activation of astrocyte hemichannels, which could have significant consequences on eCB-dependent synaptic plasticity.